Viral vaccines

ABSTRACT

A mutant virus for use as a vaccine, wherein the genome of the virus is defective in respect of a gene essential for the production of infectious virus. In one aspect the mutant virus is capable of protecting a susceptible species immunized therewith against infection by the corresponding wild-type virus. In another aspect, the mutant virus acts as a vector for an immunogenic protein derived from a pathogen and which is encoded by foreign DNA incorporated in the mutant virus. The mutant virus can be produced in a recombinant host cell which expresses a gene complementing the defect. The mutant virus is preferably infectious for the host to be protected, but the defective gene allows expression in the infected host of at least some of the vital genes, which can provoke a cell-mediated immune response.

This is a continuation of application Ser. No. 08/030,073 filed May 20,1993, now abandoned.

The present invention relates to vital vaccines. In particular, itrelates to genetically engineered mutant viruses for use as vaccines;vaccines comprising the mutant viruses; recombinant cell; and to methodsrelating to the production of vaccines.

Viral vaccines are traditionally of two sorts. The first sort are`killed` vaccines, which are virus preparations which have been killedby treatment with a suitable chemical such as beta-propiolactone. Thesecond type are live `attenuated` vaccines, which are viruses which havebeen rendered less pathogenic to the host, either by specific geneticmanipulation of the virus genome, or, more usually, by passage in sometype of tissue culture system. These two types of vaccine each havetheir own disadvantages. Since killed vaccines do not replicate in thehost, they must be administered by injection, and hence may generate aninappropriate kind of immune response. For example the Salk vaccine, akilled preparation of poliovirus, produces an immunoglobulin (Ig) Gantibody response, but does not stimulate the production of IgA in thegut, the natural site of primary infection. Hence this vaccine, thoughit can protect the individual from the neurological complications ofpoliomyelitis, does not block primary infection, and so does not confer"herd immunity". In addition, killed viruses, do not enter and replicateinside host cells. Hence any beneficial immunological response tonon-structural proteins produced during replication is not available.They also cannot stimulate the production of cytotoxic T cells directedagainst virus antigens. "Dead" antigens can be picked up by antigenpresenting cells and presented to T cells. However, the presentationoccurs via MHC Class II molecules and leads to stimulation of T helpercells. In turn, the T helper cells help B cells to produce specificantibody against the antigen. In order to stimulate the production ofcytotoxic T cells, virus antigens must be processed through a particularpathway inside the infected cell, and presented as broken-up peptidefragments on MHC Class I molecules. This degradation pathway is thoughtto work most effectively for proteins that are synthesised inside theinfected cell, and hence only virus that enters host cells and expressesimmunogenic vital protein is capable of generating virus-specificcytotoxic T cells. Therefore, killed vaccines are poor inducers ofcellular immunity against virus infection. From this point of view, liveattenuated vaccines are more satisfactory.

To date, live attenuated viruses have been made by deleting aninessential gene or partly damaging one or more essential genes (inwhich case, the damage is such that the genes are still functional, butdo not operate so effectively). However, live attenuated viruses oftenretain residual pathogenicity which can have a deleterious effect on thehost. In addition, unless the attenuation is caused by a specificdeletion, there remains the possibility of reversion to a more virulentform. Nevertheless, the fact that some viral protein production occursin the host means that they are often more effective than killedvaccines which cannot produce such vital protein.

Live attenuated viruses, as well as being used as vaccines in their ownright, can also be used as `vaccine vectors` for other genes, in otherwords carriers of genes from a second virus (or other pathogen) againstwhich protection is required. Typically, members of the pox virus familyeg. vaccinia virus, are used as vaccine vectors. When a virus, is usedas a vaccine vector, it is important that it causes no pathogeniceffects. In other words it may need to be attenuated in the same waythat a simple virus vaccine is attenuated. The same disadvantages asthose described above, therefore apply in this case.

It has been found possible to delete a gene from a vital genome andprovide a so-called `complementing` cell which provides the virus withthe product of the deleted gene. This has been achieved for certainviruses, for example adenoviruses, herpesviruses and retroviruses. Foradenoviruses, a human cell line was transformed with fragments ofadenovirus type 5 DNA (Graham, Smiley, Russell & Nairn, J. Gen. Virol.,36,59-72, 1977). The cell line expressed certain viral genes, and it wasfound that it could support the growth of virus mutants which had thosegenes deleted or inactivated (Harrison, Graham & Williams, Virology 77,319-329, 1977). Although the virus grew well on this cell line (the`complementing cell line`) and produced standard vital particles, itcould not grow at all on normal human cells. Cells expressing theT-antigen-encoding region of the SV40 virus genome (a papovavirus) havealso been shown capable of supporting the replication of virusesspecifically deleted in this region (Gluzman, Cell, 23,182-195, 1981)-For herpes simplex virus, cell lines expressing the gB glycoprotein (Caiet al, J. Virol. 62,714-721, 1987) the gD glycoprotein (Ligas andJohnson, J. Virol. 62,1486, 1988) and the Immediate Early protein ICP4(Deluca et al., J. Virol., 56,558, 1985) have been produced, and thesehave been shown capable of supporting the replication of viruses withspecifically inactivated copies of the corresponding genes.

The present invention provides a mutant virus for use as a vaccine, inwhich a vital gene encoding a protein which is essential for theproduction of infectious virus has been deleted or inactivated; andwherein said virus can be grown. in a cell which has a heterologousnucleotide sequence which allows said cell to express the essentialprotein encoded by said deleted or inactivated viral gene.

The present invention also provides a vaccine which comprises a virus asdescribed above, together with one or more excipients and/or adjuvants.The viral genome may itself provide the immunogen, or it may contain aheterologous gene insert expressing the immunogenic protein.

The present invention also provides a complementing cell transfectedwith an attenuated virus as described above, for use in the preparationof a vaccine.

The present invention also provides a method which comprises the use ofa virus as described above in the preparation of a vaccine for thetherapeutic or prophylactic treatment of a disease.

The present invention also provides a method for the production of avaccine which comprises: culturing a cell infected with a virus having adeleted or inactivated vital gene encoding a protein which is essentialfor the production of infectious virus, and wherein the host cell has aheterologous nucleotide sequence comprising said vital gene and which isable to express the essential protein encoded by said gene; harvestingthe virus thus produced, and using it in a vaccine.

The virus may be derived from herpes simplex virus (HSV) in which, forexample, the gene encoding glycoprotein H (gH) has been inactiveted ordeleted. The mutant virus may also comprise a heterologous sequenceencoding an immunogen derived from a pathogen. The host cell willsuitably be a recombinant eukaryotic cell line containing the geneencoding HSV glycoprotein H. As another example the virus may be derivedfrom an orthopox virus, for example, vaccinia virus, which again maycomprise a heterologous sequence encoding an immunogen derived from apathogen.

This invention shows a unique way of combining the efficacy and safetyof a killed vaccine with the extra immunological response induced by thein vivo production of viral protein by the attenuated vaccine. Inpreferred embodiments it comprises two features. Firstly, a selectedgene is inactivated within the virus genome, usually by creating aspecific deletion. This gene will be involved in the production ofinfectious virus, but preferably not preventing replication of the vitalgenome. Thus the infected cell can produce more viral protein from thereplicated genetic material, and in some cases virus particles may beproduced, but these would not be infectious. This means that the vitalinfection cannot spread from the site of inoculation.

A second feature of the invention is a cell which provides the viruswith the product of the deleted gene, thus making it possible to growthe virus in tissue culture. Hence, although the virus lacks a geneencoding an essential protein, if it is grown in the appropriate hostcell, it will multiply and produce complete virus particles which are tooutward appearances indistinguishable from the original virus. Thismutant virus preparation is inactive in the sense that it has adefective genome and cannot produce infectious virus in a normal host,and so may be administered safely in the quantity required to generatedirectly a humoral response in the host. Thus, the mutant virus need notbe infectious for the cells of the host to be protected and merelyoperates in much the same way as a conventional killed or attenuatedvirus vaccine. However, preferably the immunising virus is itself stillinfectious, in the sense that it can bind to a cell, enter it, andinitiate the vital replication cycle and is therefore capable ofinitiating an infection within a host cell of the species to beprotected, and producing therein some virus antigen. There is thus theadditional opportunity to stimulate the cellular arm of the host immunesystem.

The deleted or inactivated gene is preferably one involved as late aspossible in the vital cycle, so as to provide as many vital proteins aspossible in vivo for generating an immunogenic response. For example,the gene may be one involved in packaging or some other post-replicativeevent, such as the gH glycoprotein of HSV. However, the selected genemay be one involved in the vital genome replication, and the range ofproteins expressed in vivo will depend upon the stage at which that geneis normally expressed. In the case of human cytomegalovirus (HCMV) theselected gene may be one (other than the Immediate Early gene) thateffectively prevents vital genome replication in vivo, since theImmediate Early gene which is produced prior to vital genome replication(and indeed is essential for it) is highly immunogenic.

This invention can be applied to any virus where one or more essentialgene(s) can be identified and deleted from or inactivated within thevirus genome. For DNA viruses, such as Adeno, Herpes, Papova, Papillomaand Parvo viruses, this can be achieved directly by (i) the in vitromanipulation of cloned DNA copies of the selected essential gene tocreate specific DNA changes; and (ii) re-introduction of the alteredversion into the virus genome through standard procedures ofrecombination and marker rescue. The invention however, is alsoapplicable to RNA viruses. Techniques are now available which allowcomplementary DNA copies of a RNA virus genome to be manipulated invitro by standard genetic techniques, and then converted to RNA by invitro transcription. The resulting RNAs may then be re-introduced intothe virus genome. The technique has been used to create specific changesin the genome of both positive and negative stranded RNA viruses, e.g.poliovirus (Racaniello and Baltimore, Science, 214, 916-919, 1981) andinfluenza virus (Lutyes et al., Cell, 59, 1107-1113, 1989).

In theory, any gene encoding an essential protein should be a potentialtarget for this approach to the creation of attenuated viruses. Inpractice however, the selection of the gene will be driven by a numberof considerations.

1. The gene should preferably be one which is required later ininfection. Thus replication of the attenuated virus is not interruptedin the early phase. This means that most and possibly all other virusantigens will be produced in the infected cell, and presented to thehost immune system in conjunction with host cell MHC class 1 molecules.Such presentation leads to the development of cellular immunity againstvirus infection through the production of cytotoxic T cells. Thecytotoxic T cells can recognise these antigens, and therefore kill virusinfected cells. It is possible that the deleted gene could represent onewhich is not required at all for virus assembly, but is necessary forthe assembled virus to be able to infect new cells. An example of such aprotein is the HSV gH protein. In the absence of this protein, HSVvirions are still produced, but they are non-infectious.

2. Ideally, the product of the selected gene should not, on its own, betoxic to the eukaryotic cell, so that a complementing cell can beproduced relatively easily. This however is not an absolute requirement,since the gene may be placed under the control of an inducible promoterin the complementing cell, such that its expression may be switched ononly when required.

The nature of the mutation created in the target gene is also a matterof choice. Any change which produces a non-functional gene product issatisfactory, as long as the risk of reversion to a wild type structureis minimised. Such changes include interruption of the target withextraneous sequences and creation of specific deletions. The mostsatisfactory strategy for a vaccine to be used as a therapeutic and/orprophylactic however, would be one where a deletion is made thatencompasses the entire sequence to be introduced into the complementingcell. The approach minimises the risk of regenerating wild type virusthrough recombination between the virus and cell DNA in thecomplementing cell.

Although there are several examples of combinations of specificallyinactivated viruses and complementing cells, (see earlier discussion),to date, these have been used either for basic research on the virus,or, as in the case of retroviruses, to make a safer vector for producingtransgenic animals. They have not been used for vaccine purposes, and tothe applicants knowledge no suggestion of this kind of use has beenproposed.

As well as using such an inactivated virus/complementing cellcombination to produce safe vaccines against the wild-type virus, thisinvention also deals with the use of the same system to produce safevital vectors for use as vaccines against foreign pathogens.

An example of such a vector is one based on HSV. The HSV genome is largeenough to accommodate considerable additional genetic information andseveral examples of recombinant HSV viruses carrying and expressingforeign genetic material have been described (e.g. Ligas and Johnson, J.Virol. 1988, op. cit.). Thus a virus with a deletion in an essentialvirus gene as described above, and also carrying and expressing adefined foreign gene, could be used as a safe vector for vaccination togenerate an immune response against the foreign protein.

A particular characteristic of HSV is that it may become latent inneurones of infected individuals, and occasionally reactivate leading toa local lesion. Thus an HSV with a deletion in an essential virus geneand expressing a foreign gene could be used to produce deliberatelylatent infection of neurones in the treated individual. Reactivation ofsuch a latent infection would not lead to the production of a lesion,since the virus vector would be unable to replicate fully, but wouldresult in the onset of the initial part of the virus replication cycle.During this time expression of the foreign antigen could occur, leadingto the generation of immune response. In a situation where the deletedHSV gene specified a protein which was not needed for virus assembly,but only for infectivity of assembled virions, such a foreign antigenmight be incorporated into the assembled virus particles, leading toenhancement of its immunogenic effect. This expression of the foreigngene and incorporation of its protein in a viral particle could ofcourse also occur at the stage where the mutant virus is first producedin its complementing host, in which case the mutant virus when used as avaccine could present immediately the foreign protein to the speciesbeing treated.

In another example, vaccinia virus, a poxvirus, can carry and expressgenes from various pathogens, and it has been demonstrated that theseform effective vaccines when used in animal experimental systems. Thepotential for use in humans is vast, but because of the known sideeffects associated with the widespread use of vaccinia as a vaccineagainst smallpox, there is reluctance to use an unmodified vacciniavirus on a large scale in humans. There have been attempts to attenuatevaccinia virus by deleting non-essential genes such as the vacciniagrowth factor gene (Buller, Chakrabarti, Cooper, Twardzik & Moss, J.Virology 62,866-874, 1988). However, such attenuated viruses can stillreplicate in vivo, albeit at a reduced level. No vaccinia virus with adeletion in an essential gene has yet been produced, but such a virus,deleted in an essential gene as described above, with its complementingcell for growth, would provide a safer version of this vaccine vector.

A further advantage of this general strategy for immunisation againstheterologous proteins is that it may be possible to perform multipleeffective vaccinations with the same virus vector in a way not possiblewith conventional live virus vectors. Since a standard live virusvaccine probably relies for its efficacy on its ability to replicate inthe host animal through many cycles of infection, its usefulness will beseverely curtailed in an individual with immunity against that virus.Thus a second challenge with the same virus, whether to provide abooster immunisation against the same protein, or a new response againsta different protein, is likely to be ineffective. Using a virus vectorwith a deletion in an essential gene however, where multi-cyclereplication is not desired or required, the events leading to effectiveimmunisation will occur very soon after immunisation. The dose of themutant virus can be relatively large (since it should be completelysafe), and it is therefore unlikely that these early events will beblocked by the host immune response, which will require some time to bemobilised completely.

Although we have referred above to a mutant virus being defective in anessential gene, and optionally containing a gene for an immunogenicpathogen protein, the mutant could be defective in more than oneessential gene, and/or contain more than one immunogenic pathogenprotein gene. Thus, the mutant virus might include the gene for HIV gp120, to act as a vaccine in the manner suggested above, and also thegene for the HIV gag protein to be expressed within the vaccinated hostand presented at the surface of the host cell in conjunction with MHC-Ito stimulate a T-cell response in the host.

In order that the invention is more clearly understood, it will befurther described by way of example only, and not by way of limitation,with reference to the following figures in which:

FIG. 1 illustrates the production of plasmid pGH1;

FIG. 2 illustrates the production of plasmid pGH2;

FIG. 3a shows the pair of Complementary oligonucleotides (SEQ ID NO:1,SEQ ID NO:2) used to generate the plasmid pSP64Ta;

FIG. 3b illustrates the production of plasmid pSP64TA;

FIG. 4a shows the two oligonucleotides (SEQ ID NO:4, SEQ ID NO:5) usedto generate the plasmid pCMVIEP;

FIG. 4b illustrates the plasmid pCMVIEP;

FIG. 5 illustrates the plasmid pCMVlacZ; and

FIG. 6 illustrates the plasmid pGH3.

FIG. 7 illustrates the strategy for construction of plasmid pGH-120.

HERPES SIMPLEX VIRUS DELETED IN GLYCOPROTEIN H (GH-HSV )

Herpes simplex virus (HSV) is a large DNA virus which causes a widerange of pathogenic symptoms in man, including recurrent facial andgenital lesions, and a rare though often fatal encephalitis. Infectionwith this virus can be controlled to some extent by chemotherapy usingthe drug Acyclovir, but as yet there is no vaccine available to preventprimary infection. A difficulty with vaccination against HSV is that thevirus generally spreads within the body by direct transfer from cell tocell. Thus humoral immunity is unlikely to be effective, sincecirculating antibody can only neutralise extracellular virus. Of moreimportance for the control of virus infection, is cellular immunity, andso a vaccine which is capable of generating both humoral and cellularimmunity, but which is also safe, would be a considerable advantage.

A suitable target gene for inactivation within the HSV genome is theglycoprotein H gene (gH). The gH protein is a glycoprotein which ispresent on the surface of the virus envelope. This protein is thought tobe involved in the process of membrane fusion during entry of the virusinto the infected cell. This is because temperature sensitive virusmutants with a lesion in this gene are not excreted from virus infectedcells at the non-permissive temperature (Desai et al., J. Gert. Virol.69, 1147-1156, 1988). The protein is expressed late in infection, and soin its absence, a considerable amount of virus protein synthesis maystill occur.

All genetic manipulation procedures are carried out according tostandard methods described in "Molecular Cloning", A Laboratory Manual,eds. Sambrook, Fritsch and Maniatis, Cold Spring Harbor LaboratoryPress, 1989.

A. Generation of a Cell Line Expressing the HSV gH Gene

The gH gene is present in the Unique Long region (U_(L)) of the HSV type1 genome, between nucleotides 46382 and 43868 (McGeoch et al, J. Gen.Virol. 69, 1531-1574, 1988). A cloned copy of this gene is availablewithin the plasmid pAF2. This plasmid was produced by excising aBglII-Xhol fragment, encompassing the complete gH coding sequence, fromthe plasmid pTZgH, and cloning it into the BgIII site of plasmid pSP64Tas described (Gompels and Minson, J. Virol., 63, 4744-4755, 1989). AHindIII fragment containing the promoter sequence for the glycoprotein D(gD) gene (extending from nucleotides -392 to +11, with respect to thestart of the gD gene) is then excised from the plasmid pSVD4 (Everett,Nucl. Acids Res., 11, 6647-6667, 1983), and cloned into the uniqueHindIII site of pAF2 to generate pGH1 (FIG. 1) such that the promotersequence is in the correct orientation to drive expression of the gHgene. Thus this plasmid contains the complete gH coding sequence underthe control of the HSV type 1 gD gene promoter. This plasmid is thenpurified and then co-transfected into Veto cells with the plasmid pNEO(Pharmacia LKB Biotechnology Inc.) using the standard calcium phosphateco-precipitation technique (Graham and Van der Eb, Virology 52, 456-467,1973). Veto cells which have acquired resistance to neomycin are thenselected by passage of the cells in the drug G418, and colonies of thesecells cloned by limiting dilution. These neomycin resistant cells arethen amplified in tissue culture, and samples are then infected with HSVtype 2 virus. Infection with the HSV type 2 virus has the effect ofinducing transcription from the type 1 gD promoter present in thecomplementing cell genome, and so of stimulating production of the type1 gH protein in the complementing cell. Lysates of the infected cellsare then screened for expression of the gH protein by western blotting,using a polyclonal antiserum known to recognise specifically the type 1gH protein (Desai et al., 1988 op cit.). Cells which express therequired protein are then retained and frozen stocks prepared. Thismaterial represents the gH+ complementing cell line.

B. Production of HSV Type 1 Virus with an Interrupted gH Gene

A 6432 base pair BgIII fragment containing the coding sequence of gHtogether with HSV flanking sequences is excised from the plasmid pUG102(Gompels and Minson, Virology 153, 230-247, 1986) and cloned into theplasmid pAT153 (Twigg and Sherrat, Nature, 283,216, 1980) to generatepGH2 (FIG. 2). This plasmid is digested with PvuII which cuts onlywithin the gH coding sequence at two positions (nucleotides 44955 and46065 according to the numbering scheme of McGeoch et al, 1988, opcit.), and the larger of the two fragments purified. A fragment of DNAconsisting of the complete B-galactosidase gene from E coli downstreamof the Immediate Early gene promoter from Cytomegalovirus (CMV) is thenprepared by the following procedure. First of all a pair ofcomplementary oligonucleotides (SEQ ID NO:1,SEQ ID NO:2) (shown in FIG.3a) are annealed and ligated with BglII-digested, phosphatase-treatedpSP64T (Krieg and Melton, Nucl. Acids Res. 12, 7057-7071, 1984) togenerate the plasmid pSP64Ta as shown in FIG. 3b (SEQ ID NO:3). Theadded linker (SEQ ID NO:3) also includes the initiation codon and firstthree codons of the B-galactosidase gene (lacZ) of E.coli. Next the"core region" of the Immediate Early gene promoter of CMV is amplifiedfrom plasmid pUG-H1 (Gompels and Minson, 1989, op cit.) by thePolymerase Chain Reaction technique (PCR-Molecular Cloning, ed. Sambrooket al., op cit.) using the two oligonucleotides (SEQ ID NO:4; SEQ IDNO:5) shown in FIG. 4a, which correspond to sequences from -302 to -288(SEQ ID NO:4), and from -13 to -36 (SEQ ID NO:5) respectively (numberedin relation to the start of the CMV Immediate Early gene as described byAkrigg et al., Virus Research, 2, 107-121, 1985). These oligonucleotides(SEQ ID NO:4, SEQ ID NO:5) also contain, at their 5' ends, sites for therestriction enzyme HindlII, and in the case of the oligonucleotideannealing upstream of the promoter SEQ ID NO:4), an additional SmaIsite. The PCR-amplified product DNA is then digested with HindIII, andcloned into HindIII-digested pSP64Ta, to generate the plasmid pCMVIEP(FIG. 4b). Finally, a DNA fragment containing a complete copy of theE.coli B-galactosidase gene, lacking only the extreme 5' end of thecoding sequence, is isolated by digestion of the plasmid pSC8(Chakrabarti et al., Mol. Cell. Biol.,5,3403-3409, 1985) with BamHI, andcloned into the unique BgIII site of pCMVIEP to generate pCMVlacZ (FIG.5). A fragment of DNA containing the B-galactosidase gene under thecontrol of the CMV IE promoter is then isolated by digestion of pCMVlacZwith SmaI, and ligated with the purified PvuII fragment of pGH2described above, to generate pGH3, which consists of a copy of the gHgene interrupted by a functional B-galactosidase gene (FIG. 6). The nextstep is to replace the wild type gH gene in the HSV genome with thisinterrupted version, and this is done by allowing recombination betweenHSV DNA and plasmid pGH3, followed by selection of those viruses whichhave acquired a functional B-galactosidase gene. Plasmid pGH3 DNA istherefore cotransfected into cells expressing the gH gene (the gH+complementing cell line described in section A) along with purified HSVDNA isolated from purified HSV virions (Killington and Powell, In"Techniques in Virology: A practical Approach" (ed. B. W. J. Mahy) pp.207-236, IRL Press, Oxford (1985)) by the standard calcium phosphateprecipitation technique (Graham and Van der Eb, 1973, op cit.) Theprogeny HSV virus produced from this transfection experiment is thenplated on monolayers of gH+ complementing cells by standard plaqueassay, using an agar overlay, in the presence of5-bromo-chloro-3-indolyl-β-D-galactoside (X-gal), a chromogenicsubstrate which is converted to a blue substance by the enzymeβ-galactosidase. Thus plaques resulting from infection by virus genomescontaining and expressing the B-galactosidase gene will appear blue.These virus genomes should therefore carry an interrupted version of thegH gene. Virus is recovered from these plaques by picking plugs of agarfrom the appropriate part of the plate, and virus stocks preparedthrough growth of virus in the gH+ complementing cell line. Theseviruses, since they bear non-functional versions of the gH gene, shouldbe unable to form plaques on cells which do not contain and express anendogenous functional copy of the gH gene, and so to confirm this, asample of the virus is assayed for its ability to form plaques on wildtype Vero cell monolayers in comparison with the gH-complementing cells.Finally, virus DNA is prepared from these stocks, and checked for theexpected DNA structure around the gH gene by Southern blotting. Afterconfirmation of the correct genetic structure, a large stock of the gHgene-deficient virus is then prepared by inoculation of a sample of thevirus into a large-scale culture of the gH+ complementing cell line(multiplicity of infection=0.01), and three days later, the infectedcells are harvested. The infected cells are disrupted by sonication inorder to release the cell-associated virus, and the total sonicatedmixture stored at -70° as the virus master stock. The titre of the virusstock is then established by plaque assay on the gH+ complementing cellline. Samples of this virus stock are then used to prepare workingstocks as before, and these working stocks are then used to infectlaboratory animals as described below.

C. Studies on the Protective Effect of gH-HSV Compared to Heat KilledVirus

In order to assess the host immunological response to this virus,challenge experiments were conducted in mice according to theexperimental plan described below.

The protective effect of a live gH⁻ virus preparation was compared withan inactivated preparation of wild type (WT) virus (strain SC16) asfollows,

Preparation of Inactivated Wild Type Virus for Vaccination

HSV type 1 (strain SC16) was grown by low multiplicity infection(0.01pfu/cell ) of Vero cells. After three days, the virus washarvested, and cytoplasmic virus recovered by Dounce homogenisation.Nuclei were removed by centrifugation at 500×g for 15 min, and the viruswas recovered from the supernatant by centrifugation on to a 40% sucrosecushion at 12K for 60 min Beckman Sw27 rotor. The banded virus wasdiluted, pelleted and purified by sucrose gradient centrifugation(Killington and Powell, 1985, op. cit.). The virus band was harvestedfrom the gradient, and the virus recovered by centrifugation. Virus wasresuspended in phosphate-buffered saline (PBS), assayed for infectivityby plaque titration on baby hamster kidney (BHK) cells, and the particlecount determined by electron microscopy. The particle:infectivity ratioof the preparation was 110 particles/pfu. The virus was diluted to2.5×10¹⁰ pfu/ml in PBS, and inactivated by treatment withβ-propiolactone for 60 min at 20° C. Aliquots were then stored at -70°C.

Preparation of Live gH⁻ Virus for Vaccination.

This material was prepared as described for the wild type virus, exceptthat the virus was grown in the gH+ complementing cell line containingand expressing the HSV type 1 gH gene, and it was not inactivated bytreatment with β-propiolactone. The particle: infectivit ratio of thispreparation was 150:1. The concentration of this preparation wasadjusted to 2.5×10¹⁰ pfu/ml, and aliquots were stored in PBS at -70° C.

Vaccination Protocol

4 week-old female balb/C mice (purchased from Tucks U.K. Ltd) werevaccinated with various doses of inactivated WT virus or live gH- virusin 2 μl volumes of phosphate-buffered saline by droplet application andneedle scarification of the right ear as follows:

    ______________________________________                                        Group A          Control - no virus                                           Group B          5 × 10.sup.4 pfu virus vaccine                         Group C          5 × 10.sup.5 pfu virus vaccine                         Group D          5 × 10.sup.6 pfu virus vaccine                         Group E          5 × 10.sup.7 pfu virus vaccine                         ______________________________________                                    

After 14 days, all mice were challenged by similar inoculation of theleft ear with 2×10⁶ pfu HSV-1 strain SC16 (wild type virus). Mice werekilled after 5 days and assayed for virus infectivity in the left earand left cervical ganglia cII, cIII and cIV (combined). For latencystudies, other vaccinated and challenged animals were killed after 1month, and tested for latent infection by dissecting out the cII, cIIIand cIV ganglia. These were incubated in medium for five days thenhomogenised and assayed for the presence of infectious virus by standardplaque assay. All the following results are expressed as pfu/organ.

                  TABLE 1                                                         ______________________________________                                        Titre of challenge virus present during the acute                             phase of infection after vaccination with live gH- virus                                 Virus titre - log.sub.10 pfu (WT SC16)                             Mouse no.    Ears   mean     cervical ganglia*                                                                       mean                                   ______________________________________                                        group A                                                                             1          4.2           3.3                                                  2          4.2    4.3    3.4       3.4                                        3          4.6           3.4                                                  4          4.3           3.4                                            group B                                                                             1          3.4           1.5                                                  2          none   0.85   2.4       1.8                                        3          none          2.0                                                  4          none          1.5                                            group C                                                                             1          none   --     none      --                                         2          none          none                                                 3          none          none                                                 4          none          none                                           group D                                                                             1          none   --     none      --                                         2          none          none                                                 3          none          none                                                 4          none          none                                           group E                                                                             1          none   --     none      --                                         2          none          none                                                 3          none          none                                                 4          none          none                                           ______________________________________                                         *Pooled cervical ganglia cII, cIII and cIV                               

                  TABLE 2                                                         ______________________________________                                        Titre of challenge virus present during the                                   acute phase of infecton after vaccination with                                inactivated WT HSV-1                                                                     Virus titre - log.sub.10 pfu (WT SC16)                             Mouse no.    Ears   mean     cervical ganglia*                                                                       mean                                   ______________________________________                                        group A                                                                             1          5.7           2.6                                                  2          4.4    5.2    2.3       2.3                                        3          5.7           2.1                                            group B                                                                             1          4.2           1.9                                                  2          3.6    3.8    3.1       1.2                                        3          3.5           none                                                 4          3.8           none                                           group C                                                                             1          none   2.0    none      --                                         2          2.5           none                                                 3          2.9           none                                                 4          2.7           none                                           group D                                                                             1          3.9    2.6    none      --                                         2          2.0           none                                                 3          2.0           none                                                 4          2.3           none                                           group E                                                                             1          none   --     none      --                                         2          none          none                                                 3          none          none                                                 4          none          none                                           ______________________________________                                         *Pooled cervical ganglia cII, cIII and cIV                               

                  TABLE 3                                                         ______________________________________                                        Titre of challenge virus present as latent virus                              in the cervical ganglia after vaccination with live gH-                       HSV-1                                                                                      Virus titre in-                                                               cervical ganglia*                                                mouse no.    (log.sub.10 pfu WT)                                                                       reactivation frequency                               ______________________________________                                        group A                                                                             1          5.4         5/5                                                    2          4.6                                                                3          5.0                                                                4          4.8                                                                5          5.3                                                          group B                                                                             1          none        3/4                                                    2          1.5                                                                3          5.1                                                                4          5.3                                                          group C                                                                             1          none        1/3                                                    2          none                                                               3          3.2                                                          group D                                                                             1          none        0/4                                                    2          none                                                               3          none                                                               4          none                                                         group E                                                                             1          none        0/4                                                    2          none                                                               3          none                                                               4          none                                                         ______________________________________                                         *Pooled cervical ganglia cII, cIII and cIV                               

                  TABLE 4                                                         ______________________________________                                        Titre of latent challenge virus in the cervical                               ganglia after vaccination with inactivated WT HSV-1                                        Virus titre in-                                                               cervical ganglia*                                                mouse no.    (log.sub.10 pfu WT)                                                                       reactivation frequency                               ______________________________________                                        group A                                                                             1          none        3/4                                                    2          5.0                                                                3          5.0                                                                4          5.2                                                          group B                                                                             1          3.5         3/4                                                    2          4.0                                                                3          5.5                                                                4          none                                                         group C                                                                             1          3.6         2/4                                                    2          5.1                                                                3          none                                                               4          none                                                         group D                                                                             1          none        1/4                                                    2          4.8                                                                3          none                                                               4          none                                                         group E                                                                             1          none        0/4                                                    2          none                                                               3          none                                                               4          none                                                         ______________________________________                                         *Pooled cervical ganglia cII, cIII and cIV                                    (p.f.u = plaque forming units; gH is a virus with a defective gH gene).  

These results show the titre of the challenge virus wt SC16 present inthe ears and cervical ganglia during the acute phase of infection. Thus,a low titre indicates good effectiveness of the vaccination regimen withgH- virus whereas a higher titre, indicates poorer effectiveness. It isclear from the results that vaccination with live gH- HSV virus is verymuch more effective than an equivalent amount of inactivated WT virus.With the inactivated preparation, a dose of 5×10⁷ pfu was required toprevent challenge virus replication in the ear, whereas with the livegH- virus, 100-1000 fold less virus was required. Live gH- virusvaccination with 5×10⁵ pfu and over, was also able to block replicationof the challenge virus in the cervical ganglia during the acute phase ofinfection, and furthermore showed a clear protective effect against theestablishment of latent infection in the cervical ganglia.

HSV Lacking the gH Gene as a Vector for Immunisation Against a ForeignAntigen: Introduction of the gp120 Gene of SIVmac Strain 142 into theGenome of gH-HSV Virus

Viruses with deletions in essential genes may, as described above, beused as safe vectors for the delivery of foreign antigens to the immunesystem, and the gH- HSV virus described above provides a suitableexample of a such a vector. This virus could be used to express anydesired foreign antigen, but a particularly attractive possibility wouldbe the major antigenic proteins of the AIDS virus human immunodeficiencyvirus (HIV). Thus these sequences would be inserted into the gH- HSVgenome in a way that would ensure their expression during infection ofnormal cells (i.e. non-complementing cells) by the recombinant virus.Infection of an individual with such a virus could lead to a latentinfection which, from time to time upon reactivation, would lead to aburst of production of the foreign antigen, resulting in stimulation ofthe immune response to that protein.

Since studies to test this approach directly in humans are not feasibleat present, as an initial stage, the approach may be tested in monkeysusing the Simian AIDS virus SIV_(mac) (Simian immunodeficiency virusisolated from macaques). A suitable SIV gene for this purpose is thatencoding the gp120 protein, one of the major antigenic targets for thisvirus. This gene is therefore introduced into the gH- HSV genome, andthe efficacy of this virus as a vaccine to protect monkeys againstchallenge with SIV assessed.

The SIV gp120 gene is first of all cloned next to the cytomegalovirus IEcore promoter (Gompels and Minson, 1989, op. cit.), and subsequently aDNA cassette consisting of the gp120 gene and the upstream CMV promoteris cloned into plasmid pGH2 (FIG. 2). The resulting plasmid is thenco-transfected into the gH+ complementing cell line along with DNApurified from the gH- HSV, and recombinant virus which has acquired thegp120 gene in place of the β-galactosidase gene present in the gH- HSVvirus is isolated by screening for interruption of the β-galactosidasegene.

A. Construction of Plasmid for Recombination of the SIV gp120 CodingSequence into the HSV Genome

The overall scheme for this procedure is shown in FIG. 7. A SacIrestriction enzyme fragment (corresponding to bases 5240-8721) isexcised from a cloned DNA copy of the SIV genome (Chakrabarti et al.,Nature 328, 543 (1987), and cloned into the SacI site of plasmid pUC118(Viera and Messing, Methods in Enzymology, 153, 3, 1987) in order togenerate plasmid pSIV1 which may be converted to single stranded DNA formanipulation by site directed mutagenesis. This DNA region, whichincludes the SIV env gene (lying between 6090-8298) is then altered bysite directed mutagenesis (Brierley et al., Cell 57, 537, 1989) tointroduce a restriction enzyme site for the enzyme EcoRV at positions6053-6058 using the synthetic oligonucleotide (SEQ ID NO:6)

    5'GAAGAAGGCTATAGCTAATACAT.

A second EcoRV site is then introduced at position 7671-7676 within theSIV env gene corresponding to the cleavage site between the gp120 andgp40 domains of the env gene sequence, using the syntheticoligonucleotide (SEQ ID NO:7)

    5'CAAGAAATAAACTATAGGTCTTTGTGC

to generate the plasmid pSIV2. A DNA fragment (1617 base pairs)corresponding to the gp120 portion of the SIV env gene is then preparedby digestion of SIV2 with EcoRV.

The core region of the CMV immediate early gene promoter is obtainedfrom the plasmid pUG-H1 (Gompels and Minson, 1989, op cit.) by the PCRtechnique using the following two synthetic oligonucleotides (SEQ IDNO:8, SEQ ID NO:9).

    __________________________________________________________________________    upstream primer (SEQ ID NO:8)                                                 5' ATC  GAATTC  CTATAG CCTGGCATTATGCCCAGTACATG                                        EcoRI EcoRV                                                           downstream primer (SEQ ID NO:9)                                               5'TCA AAGCTT CTATAG CCCGGGGAGCTCTGATTATATAGACCTCCC                                   HindIII                                                                              EcoRV  SmaI                                                     __________________________________________________________________________

The product of this reaction is then cleaved with the enzymes EcoRI andHindIII to generate a DNA fragment which is then cloned into EcoRI-andHindIII-digested plasmid pUC118 to generate the plasmid pCMVIE2 whichhas a unique SmaI site located just downstream of the CMV promotersequence. The EcoRV fragment containing the SIV_(mac) gp120 codingsequence prepared as described above, is then cloned into this SmaIsite, and plasmid pSIV3, with the SIV coding region oriented correctlyto allow expression of the coding sequence from the promoter, is thenselected. This plasmid is then digested with EcoRV to yield ablunt-ended DNA fragment consisting of the SIV sequence together withthe CMV promoter, which is then cloned into PvuII-digested pGH2 (FIG. 2)to produce pGH-120.

B. Construction of the SIV gp120 Carrying Recombinant gH- HSV

DNA is purified from the gH- HSV virus constructed as detailed in theprevious. section, and co-transfected into gH+ complementing cells alongwith purified pGH-120 DNA. Progeny virus isolated from this transfectionprocedure is then plated on monolayers of the gH+ complementing cellline by standard plaque assay as before using an agar overlay in thepresence of x-gal. The parental gH- virus carries a functionalβ-galactosidase gene, located within the residual gH coding sequences,and in the presence of X-gal, will form blue plaques. Recombinantviruses however, which have acquired the SIV gp120 coding sequence inplace of the β-galactosidase gene, will produce white plaques. Virus isrecovered from these white plaques by picking plugs of agar, and virusstocks prepared through growth of the virus in the gH+ complementingcell line. Virus DNA is prepared from these stocks, and checked for thepresence of the correct DNA structure around the gH gene by SouthernBlotting using appropriate probes derived from the SIV coding sequence.Finally stocks of the virus are prepared as before for vaccinationstudies in animals.

A vaccine comprising the attenuated virus can be prepared and usedaccording to standard techniques known in the art. For example, thevaccine may also comprise one or more excipients andr adjuvants. Theeffective dose of the attenuated virus to be provided by the vaccine maybe determined according to techniques well known in the art.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 9                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: complement (5..21)                                              (D) OTHER INFORMATION: /note= "Complementary to SEQ ID                        NO:2, bases 5 to 21."                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GATCCACCATGACCATGATTA21                                                       (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: complement (5..21)                                              (D) OTHER INFORMATION: /note= "Complementary to SEQ ID                        NO:1, bases 21 to 5."                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GATCTAATCATGGTCATGGTG21                                                       (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- RNA                                                 (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note= "Start of lacZ."                                (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: complement (18..23)                                             (D) OTHER INFORMATION: /note= "BglII restriction site."                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GATCCACCATGACCATGATTAGATCT26                                                  (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 5..10                                                           (D) OTHER INFORMATION: /note= "HindIII restriction site."                     (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 10..15                                                          (D) OTHER INFORMATION: /note= "SmaI restriction site."                        (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 16..38                                                          (D) OTHER INFORMATION: /note= "CMV sequence."                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ATCAAGCTTCCCGGGCCTGGCATTATGCCCAGTACATG38                                      (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 4..9                                                            (D) OTHER INFORMATION: /note= "HindIII restriciton site."                     (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 11..33                                                          (D) OTHER INFORMATION: /note= "CMV sequence."                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TCAAAGCTTGAGCTCTGATTATATAGACCTCCC33                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 9..14                                                           (D) OTHER INFORMATION: /note= "EcoRV restriction site."                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GAAGAAGGCTATAGCTAATACAT23                                                     (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CAAGAAATAAACTATAGGTCTTTGTGC27                                                 (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 4..9                                                            (D) OTHER INFORMATION: /note= "EcoRI restriction site."                       (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 10..15                                                          (D) OTHER INFORMATION: /note= "EcoRV restriction site."                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ATCGAATTCCTATAGCCTGGCATTATGCCCAGTACATG38                                      (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 4..9                                                            (D) OTHER INFORMATION: /note= "HindIII restriction site."                     (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 10..15                                                          (D) OTHER INFORMATION: /note= "EcoRV restriction site."                       (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 16..21                                                          (D) OTHER INFORMATION: /note= "SmaI restriction site."                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TCAAAGCTTCTATAGCCCGGGGAGCTCTGATTATATAGACCTCCC45                               __________________________________________________________________________

We claim:
 1. A vaccine comprising a pharmaceutically acceptableexcipient and an effective immunizing amount of a mutant herpesvirus,said mutant herpesvirus containing a genome in which a viral geneencoding a protein which is essential for production of infectious virushas been deleted or inactivated, wherein said mutant virus is able tocause production of infectious new virus particles in a recombinantcomplementing host ceil expressing a gene which complements saidessential vital gene, but is unable to cause production of infectiousnew virus particles when said mutant virus infects a host cell otherthan said recombinant complementing host cell, for prophylactic ortherapeutic use in generating an immune response in a subject infectedtherewith.
 2. The vaccine of claim 1, wherein said essential protein isinvolved in a post-replicative event.
 3. The vaccine of claim 1, whereinsaid essential protein is not required for virus assembly, but isnecessary for the assembled virus to be able to infect new cells.
 4. Thevaccine of claim 1, which consists essentially of said pharmaceuticallyacceptable excipient and an effective immunizing amount of said mutantherpesvirus.
 5. The vaccine of claim 1, wherein the mutant herpesvirusis capable of establishing a latent infection with periodicreactivation.
 6. The vaccine of claim 1, wherein said gene which hasbeen deleted or inactivated is a glycoprotein gene.
 7. The vaccine ofclaim 1, 2, 3, 4, 5, or 6, wherein the herpesvirus is herpes simplexvirus.
 8. The vaccine of claim 6, wherein said herpesvirus is herpessimplex virus and wherein the gene which has been deleted or inactivatedis the gH gene.
 9. The vaccine of claim 8, comprising a dose containingfrom about 5×10⁴ to about 5×10⁷ pfu of said mutant virus.
 10. Thevaccine of claim 8, comprising a dose containing from about 5×10⁴ toabout 5×10⁶ pfu of said mutant virus.
 11. The vaccine of claim 8,comprising a dose containing from about 5×10⁴ to about 5×10⁵ pfu of saidmutant virus.
 12. The vaccine of claim 1, wherein the mutant herpesvirusis defective in more than one gene essential for production ofinfectious virus.
 13. A method of manufacturing a vaccine according toclaim 1, comprising the steps of:a) growing a mutant herpesvirus in arecombinant complementing host cell, wherein said mutant herpesviruscontains a genome in which a viral gene encoding a protein which isessential for production of infectious virus has been deleted orinactivated, and said recombinant complementing host cell expresses agene which complements said essential viral gene; and b) mixing theresulting virus in an effective immunizing amount with apharmaceutically acceptable excipient.
 14. The method of claim 13,wherein said essential protein is involved in a post-replicative event.15. The method of claim 13, wherein said essential protein is notrequired for virus assembly, but is necessary for the assembled virus tobe able to infect new cells.
 16. The method of claim 13, wherein themutant herpesvirus is capable of establishing a latent infection withperiodic reactivation.
 17. The method of claim 13, wherein said genewhich has been deleted or inactivated is a glycoprotein gene.
 18. Themethod of claim 13, 14, 15, 16, or 17, wherein the herpesvirus is herpessimplex virus.
 19. The method of claim 17, wherein said herpesvirus isherpes simplex virus, and wherein the gene which has been deleted orinactivated is the gH gene.
 20. The method of claim 19, wherein theeffective immunizing amount is a dose containing from about 5×10⁴ toabout 5×10⁷ Pfu of said mutant virus.
 21. The method of claim 19,wherein the effective immunizing amount is a dose containing from about5×10⁴ to about 5×10⁶ pfu of said mutant virus.
 22. The method of claim19, wherein the effective immunizing amount is a dose containing fromabout 5×10⁴ to about 5×10⁵ pfu of said mutant virus.
 23. The method ofclaim 13, wherein the mutant herpesvirus is defective in more than onegene essential for production of infectious virus.
 24. A vaccinecomprising a pharmaceutically acceptable excipient and an effectiveimmunizing mount of an infectious virus, wherein the infectious virus insaid vaccine consists essentially of a mutant herpesvirus containing agenome in which a viral gene encoding a protein which is essential forproduction of infectious virus has been deleted or inactivated, whereinsaid mutant virus is able to cause production of infectious new virusparticles in a recombinant complementing host cell expressing a genewhich complements said essential viral gene, but is unable to causeproduction of infectious new virus particles when said mutant virusinfects a host cell other than said recombinant complementing host cell,for prophylactic or therapeutic use in generating an immune response ina subject infected therewith.